Previous work on this grant focused on two complementary areas, i.e.,the function and regulation of protein kinase C-alpha (PKCalpha), which we isolated in 1993, and interactions of Syk-family PTKs with their substrates and targets in T cells. Recent studies, including our own, demonstrated that PKCtheta is a key component of the immunological synapse (IS). PKCtheta plays an essential role in mature T cell activation by integrating TCR/CD28 costimulatory signals leading to activation of the NF-kB signaling cascade and AP-1. Induction of these transcription factors, which is essential for IL-2 production and prevention of T cell anergy, is defective in anergic T cells. In view of this emerging importance of PKCtheta in T cell activation and our extensive commitment to this area, we will focus our proposed studies on the function and regulation of PKCtheta in Ag-specific T cells, with emphasis on its potential role in CD28 costimulation and anergy. First, we will elucidate the molecular and biochemical mechanism(s) that dictate the selective recruitment of PKCtheta to the IS and its activation, including the identity of putative scaffold protein(s), the role of PI3K, and the functional importance of the IS localization of PKCtheta. Second, the intermediate signaling proteins that couple PKCtheta to the NF-kB cascade are unknown. We will use Ag-specific wild type or PKCtheta-deficient T cells and Jurkat T cells in conjunction with biochemical and genetic approaches to identify these proteins, with emphasis on known components of the NF-kB and MAPK signaling cascades. Third, we will study the role of PKCtheta in transducing CD28 costimulatory signals and preventing T cell anergy by employing selective PKCtheta interfering strategies or, conversely, determining whether constitutively active PKCtheta can rescue T cell activation in CD28-deficient T cells. These studies will generate a detailed understanding of the structure-function relationship of this critical T cell enzyme, establish its role in T cell activation, costimulation, and anergy, and provide a sound basis for its future use as a drug target in T cells.